Resilience to Climate Change by Biocontrol Yeasts Against Ochratoxin A Production in Robusta Coffee

Abstract

Aspergillus carbonarius is the main producer of Ochratoxin A (OTA) in coffee. In the last few years, there has been an increasing interest in using yeast isolates as Biocontrol Agents to prevent OTA production in coffee cherries during the primary postharvest processing. Little is known about how climate change abiotic conditions of increased temperature (+2–4 °C), elevated CO₂ (existing levels of 400 vs. 1000 ppm), and increased drought stress will impact biocontrol resilience. This study examined the effect of a three-way interaction between temperature (27, 30, and 33 °C) x water activity (a_w) (0.90 and 0.95 a_w) x CO₂ level (400 vs. 1000 ppm) on the growth and OTA production of A. carbonarius and the resilience of three yeast strains’ biocontrol capacity on fresh coffee cherries. High a_w (0.95), CO₂, and temperature levels increased the production of OTA by A. carbonarius. All the yeast biocontrol strains significantly reduced A. carbonarius growth by at least 20% and OTA production by up to 85%. From the three strains used, the Meyerozyma caribbica strain (Y4) showed the best resilience to climate change, since it reduced both growth (50%) and OTA production (70%) under future scenarios of CO₂ and a_w at all temperatures tested, and should be the one selected for pilot scale experiments in Ivory Coast.

1. Introduction

In 2021/2022, 10.1 million tonnes of coffee were produced worldwide [1]. The main food safety concern linked with coffee is contamination with Ochratoxin A (OTA) [2,3]. This mycotoxin, produced by some species of the genera Aspergillus and Penicillium has both acute and potential chronic toxic effects on humans and animals [4,5]. The health impact of OTA ingestion may include mutagenicity, possible carcinogenicity (group 2B by the International Agency for Research on Cancer (IARC)), hepatoxicity, and nephrotoxicity [6]. To prevent exposure, the maximum regulated levels established by the European Union (EU) range between 3 and 5 μg/kg for roasted coffee beans and soluble coffee, respectively [7]. These limits are especially important in coffee imported from Africa, where concentrations can exceed the maximum regulation up to 10.9 µg/kg of coffee [8].

The main fungal producers of OTA on coffee sourced from Ivory Coast are A. carbonarius and A. ochraceus [8]. The growth and OTA production of these species can be influenced by environmental conditions such as temperature, water activity (a_w), and the concentration of atmospheric CO₂ [9,10]. By the year 2100, global temperatures are predicted to be 2.1–3.5 °C warmer than today, the frequency of droughts is expected to rise, and CO₂ levels could reach 1000 ppm, according to the Shared Socioeconomic Pathway (SSP) 2–4.5 [11]. The interaction between these factors has been previously studied to evaluate their influence on the growth and production of OTA by A. carbonarius on stored coffee beans from Kuwait [12]. The authors reported a 60% increase in OTA production by A. carbonarius under elevated CO₂ (1000 ppm) compared to 400 ppm CO₂ levels (at 35 °C).

The use of biocontrol microorganisms during coffee postharvest processing has shown the capacity to modulate the final OTA level [13,14]. Microorganisms such as yeast have shown promising results in the reduction of fungal growth and OTA production during coffee fermentation, thanks to their resistance to variable environmental conditions and their lack of production of spores or toxic metabolites [15,16]. Yeasts are part of coffee’s endogenous mycobiota and play a crucial role in breaking down coffee mucilage while regulating the presence of detrimental microorganisms [17,18]. Some of them, such as some of the species belonging to the genera Saccharomyces, Pichia, or Hanseniaspora are considered potential Biocontrol Agents (BCAs) thanks to their ability to reduce the growth and OTA production of ochratoxigenic fungi [19]. Recent results reported the great capacity of three yeasts identified as Rhodosporidiobolus ruineniae, Meyerozyma caribbica, and Vishniacozyma sp. to reduce fungal growth and OTA production by up to 85 and 90% in vitro [20].

The use of BCAs to reduce OTA production under the double effect of temperature and a_w has been previously explored in coffee-based media. Neves et al. [15] identified a strain of S. cerevisiae CCMA0159 capable of reducing growth and OTA production by 80 and 70%, respectively, for all toxigenic strains. Also, this strain showed good resilience by maintaining its inhibitory capacity under temperatures and a_w between 18 and 28 °C and 0.95 and 0.99 a_w [15]. However, nothing is known of these BCAs’ resilience under the triple effect of temperature, a_w, and elevated CO₂ on fresh coffee cherries. Thus, these findings could have a broader impact on global coffee production sustainability in the future under expected climatic conditions.

This study aimed to identify potential climate change-resilient BCAs against A. carbonarius and OTA production in coffee cherries. In the present work, we studied the effect of the three-way interacting climate-related abiotic factors (temperature (27–33 °C) x drought stress (0.90–0.95 a_w) x existing (400 ppm) and elevated (1000 ppm) CO₂) in fresh coffee cherries on (i) A. carbonarius growth and OTA production and (ii) the BCAs’ resilience to reduce A. carbonarius growth and OTA production under climate change scenarios.

2. Results

2.1. Effect of Climate Change on Aspergillus carbonarius Growth

The growth score was assessed on a scale of 1 to 5, where a rating of 1 denoted minimal growth, and 5 corresponded to the highest level of growth observed (Figure S1). All samples inoculated with A. carbonarius only were assigned a growth score of 5, showing a consistent capacity of A. carbonarius for growth across all the temperature, a_w, and CO₂ levels tested.

2.2. Effect of Biocontrol Agent and Abiotic Factors on Fungal Growth

Co-inoculation of A. carbonarius with the BCAs reduced fungal growth under some scenarios in comparison to the control (Figure S1). Y1 and Y2 did not impact fungal growth except at lower a_w (0.90 a_w), while the yeast Y4 decreased the growth of A. carbonarius at both a_w levels. The most important factors reducing growth were the type of strain and the a_w. Factors such as CO₂ or temperature did not impact growth. The strains Y1 and Y2 demonstrated comparable outcomes, diminishing the growth to level 3 at 0.90 a_w, with no reduction in growth observed at 0.95 a_w. The strain Y4 was the most successful in reducing A. carbonarius growth to level 2 at 0.90 a_w and to level 4 at 0.95 a_w.

2.3. Effect of Climate Change on A. carbonarius Ochratoxin A Production

Under all scenarios, A. carbonarius produced a concentration of at least 1000 µg of OTA per kg of coffee. The a_w (p < 0.05) and CO₂ (p < 0.05) factors significantly impacted the production of OTA by A. carbonarius (Table S1; Figure 1). An increase in a_w significantly increased OTA production only at 27 °C, as observed in Figure 1. Current CO₂ conditions (400 ppm CO₂) showed lower OTA production compared to elevated CO₂ (1000 ppm) at 33 °C and 0.95 a_w. The comparison of the current (400 ppm CO₂, 0.95 a_w, and 27 °C) and future (1000 ppm CO₂, 0.90 a_w, and 33 °C) combined climatic factors showed consistent OTA production by A. carbonarius in both conditions.

2.4. Efficiency of BCAs on Ochratoxin A in Climate Change Conditions

The impact of BCAs on OTA concentration was further investigated jointly with the three-way interacting climate change factors. The factors of strain, a_w, and temperature (T) and the interacting factors T* CO₂, CO₂* a_w *T, BCA*T, BCA* CO₂*a_w, and BCA* CO₂*T* a_w have a significant effect (p < 0.05) on the percentage of OTA reduction (Table S2).

In Table 1, the significance of the effect of abiotic factors on each yeast strain capacity for OTA reduction using the Fit Least Squares model was analyzed. For all strains, a_w (p < 0.05) had a significant effect on their capacity to control OTA concentration, while for strain Y4, temperature and CO₂ also had an effect on OTA reduction (p < 0.05).

As shown in Figure 2, under current CO₂ conditions and 0.90 a_w, there is a significant reduction in the OTA concentration (p < 0.05) at 33 °C by the strains Y1 and Y4 (Figure 2). At 0.95 a_w, Y4 reduces significantly (p < 0.05) OTA at 30 and 33 °C. At 33 °C and both a_w levels, Y4 reduced OTA production by at least 70%, while the Y1 and Y2 strains reduced OTA by a maximum of 70 and 40%, respectively. Under future CO₂ conditions and 0.90 a_w, there is a decrease of OTA concentration of 60 and 80% at 33 °C for Y2 and Y4, respectively. At 0.95 a_w, the reduction by the Y1 and Y4 strains is 30% at 27 °C and 40% and 60% at 33 °C for each strain, respectively. At these same conditions, Y2 shows a weaker ability to reduce OTA, of 20% and 5%, respectively, for 27 and 33 °C.

Higher CO₂ and temperature promote the reduction of OTA by the strains Y2 and Y4 at 0.90 a_w. At the same a_w but current CO₂ levels, OTA reduction is higher at 33 °C for all the strains. From all studied combinations of CO₂, temperature, and a_w, all strains reduced OTA production by at least 10%, except Y1 at 1000 ppm CO₂, 30 °C, and 0.95 a_w and Y2 at 400 ppm CO₂, 27 °C, and 1000 ppm CO₂, and 33 °C at 0.95 a_w.

3. Discussion

This study addressed for the first time how the triple effect of temperature, a_w, and CO₂ levels impact the efficiency of different yeasts as potential BCAs against growth and the production of OTA by A. carbonarius on fresh coffee beans. Previous studies only analyzed the effect of a_w and temperature (not considering the effect of CO₂) in vitro on coffee-based media, using mostly Aspergillus section Circumdati strains and without the inoculation of potential BCAs [21]. The first results from Estrada-Bahena et al. analyzed OTA contamination of coffee under a range of a_w levels between 0.108 and 0.821 during storage, concluding that at 0.82 a_w, the contamination of OTA in coffee cherries increased [22]. Oliveira et al. [23] confirmed these results with an OTA risk index of the species A. carbonarius between 2.5 and 3.5 times higher at 0.95–0.99 a_w and 25–32 °C than at 0.92 a_w and 20 °C. The authors used a model based on 11 assays performed under a variety of 0.91 to 0.99 a_w levels and temperatures of 20–38 °C.

In the present work, a_w was the most important abiotic factor increasing A. carbonarius fungal growth. Previous studies have described the optimal growth of A. carbonarius between 0.93 and 0.96 a_w in a coffee-based medium [15], which aligns with our results. Surprisingly, temperature did not influence fungal growth, while in other studies it has been positively correlated with higher A. carbonarius growth [24].

The strain Y4 was the most efficient BCA at reducing A. carbonarius growth, which is in alignment with Lopez-Rodriguez et al. [20]. Many studies have shown the effect of yeasts on A. carbonarius fungal growth [17,24,25]. Neves et al. [15] isolated S. cerevisiae CCMA 0159 from coffee beans and analyzed its growth inhibitory effect on A. carbonarius in coffee-based media. The results showed that 28 °C and 0.99 a_w were the optimum conditions for fungal growth inhibition by S. cerevisiae CCMA 0159, while in our experiment, a higher a_w did not give any advantage to the capacity of the yeasts to reduce fungal growth.

Regarding OTA production, Akbar et al. previously studied OTA production of A. carbonarius under different conditions of CO₂, temperature, and a_w on a coffee-based medium [12]. That study concluded that elevated CO₂ (1000 ppm) and temperatures (35 °C) increased OTA production by 40%. In our study, under elevated CO₂ and a_w, there was an increase in OTA production of 70% at 33 °C compared to the same conditions at ambient CO₂. This suggests that under future temperatures that are expected to rise towards 30 °C in coffee-producing areas [26], the OTA concentration may increase two-fold in coffee. Concerning a_w, higher a_w showed a correlation with slightly higher OTA production by A. carbonarius, which was significant at 27 °C. Previous studies have also shown that an a_w level above 0.95 a_w positively influenced OTA production levels by A. carbonarius [23].

The present study strain Y4 presented the best resilience, since it reduced OTA production under more scenarios. Previous studies have shown that yeast species such as S. cerevisiae can reduce the production of OTA during the confrontation against A. carbonarius on different food matrixes and under different climatic conditions. For example, Neves et al. [15] researched the strain S. cerevisiae CCMA 0159 against A. carbonarius on a coffee-based medium and showed a significant reduction of 80% in OTA production at high temperatures (28 °C) and a_w (0.99). During this investigation, expression of the OTA biosynthetic genes pks, nrps, and p450 belonging to the OTA genic cluster was reduced, potentially correlating with decreased OTA production. Similar results were reported by Cubaiu et al. [24], where S. cerevisiae strains isolated from wine were tested against A. carbonarius, demonstrating the ability to inhibit OTA production by at least 65% and reducing the expression of the pks gene as well. Complementary studies of the proposed BCAs’ effect on the downregulation of OTA biosynthetic gene expression in A. carbonarius would be needed. To our knowledge, there has been only one experimental study of a BCA against an ochratoxigenic fungi using coffee cherries as a substrate, which was conducted by De Melo Pereira et al. [27]. In this work, strains of Pichia fermentans and A. westerdijkiae were inoculated on coffee beans and incubated for 10 days at 28 °C and an a_w of 0.63 at the beginning and 0.97 a_w at the end of the incubation period. The results showed a reduction in OTA production by 88% in interaction with P. fermentans. These results align with Y4’s capacity to reduce OTA up to 80% depending on the climate scenarios. This confirms the uniqueness of our BCA efficiency trials of A. carbonarius on fresh coffee cherries, highlighting both the novel selection of biocontrol agent species—previously untested for this purpose—and the selection of the substrate.

It should be noted that the two studies previously mentioned did not monitor the experiments under controlled temperature, a_w, or CO₂ conditions. Also, there are very few publications using BCAs, such as bacteria or filamentous fungi, in coffee-based media or directly on coffee cherries [21,27]. Most of the studies applying BCAs on coffee focus on the use of Pseudomonas sp. and Trichoderma sp. for the treatment of coffee plant infections, such as coffee rust or coffee wilt disease [28,29,30,31].

Finally, we recommend the study of the proposed BCAs in confrontation with other strains of A. carbonarius and other strongly ochratoxigenic species, such as A. westerdijkiae. This would help to find out the spectrum of OTA reduction of these BCAs to tackle OTA contamination through their application in field trials.

4. Conclusions

This multifactorial study has shown that the use of BCAs such as Rhodosporidiobolus ruineniae (Y1), Vishniacozyma sp. (Y2), and Meyerozyma caribbica (Y4) in co-culture with a high OTA-producing strain of A. carbonarius on coffee cherries, could result in a reduction of up to 50 and 90% of fungal growth and OTA production, respectively. Thus, these BCAs represent an alternative to creating a more sustainable and safe coffee postharvest chain.

The efficiency of the BCAs was affected by temperature, a_w, and CO₂, resulting in a higher efficiency of OTA reduction under future conditions of elevated CO₂ and temperature and reduced a_w. M. caribbica (Y4) is proposed as the most efficient BCA, and the application of this BCA on fresh coffee cherries could reduce contamination with OTA by at least 70% under future conditions of CO₂ and a_w, representing an adaptable BCA whose optimized formulation could lead to its commercialization.

5. Materials and Methods

5.1. Coffee Sampling

Nineteen kilograms of fresh Robusta coffee cherries, which were not processed, were harvested by hand in 2020 using sterilized gloves, placed in Stomacher bags, and stored and transported at 4 °C. Upon reception, all samples were then kept at −20 °C. The coffee was cultivated in Akoupé, Côte d’Ivoire (6° 23′ north, 3° 54′ west) at an altitude of 126 m. The coffee was irradiated to eliminate the microbiota while conserving the biochemical composition, as previously described for other foodstuffs [32]. This process started by placing the coffee in a Petri dish without a cover inside a Biosafety cabinet equipped with UV-C (254.7 nm wavelength) lamps. The coffee was irradiated with UV-C for 1 h and 15 min and turned every 15 min to eliminate homogenously the native microbiota on its surface. The efficacy of the disinfection step was previously tested in fresh coffee without UV-C treatment (0 h UV-C), with 0.5 h of UV-C treatment (0.5 h UV-C), 1 h of UV-C treatment (1 h UV-C), and 1 h and 15 min (1.25 h UV-C), totaling four treatments. Afterwards, the coffee was directly plated on PDA and MRS media for fungal and bacterial growth, respectively, and incubated for 72 h. The disinfection with UV-C for 1 h and 15 min proved to be the most effective with 100% reduction of fungal and bacterial growth when compared to the non-irradiated coffee.

5.2. Yeasts and Fungal Strains

Three yeasts previously isolated from Ivorian Robusta coffee cherries were used [20]. Based on phenotypic characteristics, biochemical tests, and sequence analyses of the ITS1 and ITS2 regions and the D1/D2 domains of the 26S rRNA gene, the three yeasts were identified as Rhodosporidiobolus ruineniae (Y1), Vishniacozyma sp. (Y2), and Meyerozyma caribbica (Y4), as described by Lopez-Rodriguez et al. [20]. These strains were selected based on their ability to reduce OTA concentration by up to 90%. The native ochratoxigenic strain of A. carbonarius (D3) used in this study was isolated from Ivorian Robusta coffee cherries and identified based on phenotypic characteristics and sequence analyses of the ITS1 and ITS2 regions and the partial regions of the β-tubulin and Calmodulin genes. It was selected for its high OTA production capacity and used as a toxigenic pathogen in the present study [20].

5.3. Inoculation of Coffee Beans

The yeast strains were sub-cultured on PDB (Potato Dextrose Broth) and incubated in agitation for 48 h at 25 °C. Then, the cells were counted using a counting chamber (Neubauer improved, Paul Marienfield, GE) and suspended in a 1% (w-v) Tween^®80 solution. From this solution, a specific volume was added to reach a final adjusted concentration of 10⁶ cells/g of fresh coffee cherries. The ochratoxigenic strain was sub-cultured on PDA (Potato Dextrose Agar). After 7 days at 25 °C, the spores were removed from the colony using a sterile loop and transferred to a 1% (w-v) Tween^®80 solution. Similarly, from this solution, a specific volume was added to reach a final concentration of 10³ spores/g of fresh coffee. The final stock solution was used to modify the a_w of the cherries.

5.4. Incubation Conditions and Elevated CO₂ Treatment

Fifteen grams of fresh coffee cherries were aseptically distributed into sterile glass culture vessels of a 100 mL capacity (Magenta™, Sigma, St. Louis, MO, USA) with a vented lid (10 mm with polypropylene membrane 0.22 µm pore size) to allow gas exchange but keep the environment inside the vessel sterile. Then, 2 or 12 mL of sterile water was added to reach 0.90 or 0.95 a_w, respectively, based on the moisture adsorption curve of coffee. After adding the water, the jars were manually shaken twice to ensure good rehydration and were kept at 4 °C for 24 h for full adsorption and equilibration. The next day, the jars were inoculated with 1 mL of inoculum, either yeast and fungus, or only fungus, depending on the treatment, and equally shaken to ensure the inoculum soaked all the cherries. All treatments were run in five replicates. The jars were placed in 12 L airtight containers and incubated in a dynamic climatic chamber (SANYO Electric Co., Ltd., MLR-350H, Tokyo, Japan) at three different temperatures (27, 30, and 33 °C) and the Climate Change (CC) scenario of the Southern East region of Ivory Coast. The temperature was monitored by introducing a thermometer inside the boxes. The containers included a beaker (300 mL) of water with glycerol (vegetable origin, >99%) at 0.90 and 0.95 a_w to minimize moisture loss. They were flushed twice a day for 12 min either with current (400 ppm) or increased (1000 ppm) CO₂ concentrations by using calibrated gas cylinders (British Oxygen Company, Guildford, Surrey, UK) with a flow rate of 3 L/min to renew 3-fold the air volume of the incubation containers [33]. During the flush, the outlet hose was opened to ensure flow of the air. After 12 min, the outlet and inlet hoses were closed. After 7 days, the samples were collected and oven dried at 65 °C for 48 h. The samples were visually assessed for fungal growth, and a scoring was assigned according to Figure S1 before storage at 4 °C. The scoring system ranges from 1 to 5, with higher scores indicating increased fungal growth (Figure S1).

5.5. Ochratoxin A Quantification

The samples were processed according to Malachova et al. [34]. Briefly, the samples were ground using a laboratory blender with a stainless-steel blade (Waring, Stamford, CT, USA). Five grams of sample were extracted with 20 mL of extraction solvent (acetonitrile/water/acetic acid 79:20:1, v/v/v). The samples were extracted for 90 min using a VWR VX-2500 Multitube Vortexer (VWR, Radnor, PA, USA) at 1800 rpm speed and subsequently centrifuged for 5 min at 3000 rpm (Thermo Scientific Sorvall ST40 Centrifuge, Loughborough, UK). The extracts were transferred into glass vials and diluted with the same volume of dilution solvent (acetonitrile/water/acetic acid 20:79:1, v/v/v).

Quantification was performed using a qTRAP-LC-MS/MS 6500+ system (Exion Series) without further pre-treatment. An ACE 3-C18 column (2.1 × 100 mm, 3 µm particle size; Hichrom, Berkshire, UK) equipped with a C18 security guard cartridge (4 × 3 mm, Gemini Agilent, Oxford, UK) kept at 60 °C was used for chromatographic separation with the following gradients: Solvent A: water:methanol:acetic acid (89:10:1, v:v:v) and Solvent B: methanol:water:acetic acid (97:2:1, v:v:v), both supplemented with 5 mM ammonium acetate, which was applied for 20 min. The flow rate of the mobile phase was 0.6 mL/min, and the injection volume was set to 1 µL. MS/MS was performed in scheduled multiple reaction monitoring (MRM) with a 60 s window. Analyst version 1.6.3 (AB SCIEX™, Framingham, MA, USA) and MultiQuant version 3.0.3 (AB SCIEX™, Framingham, MA, USA) were used for data acquisition and analysis, respectively, following the method described in Malachova et al. [34].

5.6. Data Analysis

Statistical analysis was performed using the statistical package JMP^® Pro 18 (SAS Institute Inc., Cary NC, USA). Shapiro–Wilk tests were used to determine normality and Levene’s test to assess the equality of variance. The OTA production data did not follow normal distribution, and logarithmic and square root transformations did not sufficiently normalize the data for analysis. Consequently, the General Linear Model (GLM), specifically the Standard Least Squares model was selected, and the parametric Fit Least Square Method (p = 95%) was applied to analyze the effect of the factors CO₂, a_w, and T. The factors followed an LS Means Tukey HSD test to assess potential differences across treatments. The variable used in the analysis was either OTA production to assess the impact of abiotic factors on A. carbonarius’s OTA production or the percentage reduction in OTA to evaluate the effect of the biocontrol strains on A. carbonarius’s OTA production. Both variables were analyzed following the Standard Least Squares model.

Author Contributions

C.L.-R., conceptualization, formal analysis, data curation, visualization, investigation, writing—original draft, and project management; C.V.-V., writing—review and editing, conceptualization, data curation, validation, and supervision; C.S., supervision; A.F., supervision; T.G., resources; S.S.-G., supervision and funding acquisition; A.M., writing—review and editing, conceptualization, data curation, validation, supervision, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data used to support the findings of this study can be made available by the corresponding author upon request.

Acknowledgments

The authors acknowledge the great help of Monika Jodkowska for maintaining and facilitating access to the LC-MS/MS machine. The authors also acknowledge the coffee farmers from Akoupé for providing fresh coffee cherries.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

The following abbreviations are used in this manuscript:

BCA	Biocontrol Agent
OTA	Ochratoxin A
IARC	International Agency for Research on Cancer
CO2	Carbon dioxide
aw	Water activity

Footnotes

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Figures and Table

Figure 1. Effect of CO2, water activity (aw) and temperature (T, °C) on the Ochratoxin A concentration on fresh coffee inoculated with A. carbonarius. Different letters indicate significant differences (p [greater than] 0.05) between different temperatures at a specific aw and CO2 using the Fit Least Squares Method and LSMeans differences Tukey HSD. Bars represent the average and the error bars standard deviation.

Figure 2. Effect of CO2, temperature, and water activity on the % reduction in OTA concentration in fresh coffee cherries. Different letters indicate significant differences (p [greater than] 0.05) in the % reduction in OTA of each strain, using the Fit Least Squares method and Tukey HSD test. Bars represent AV ± SD.

Table 1

Summary of the statistical analysis for the effect of interacting climate-related abiotic factors on % of reduction of Ochratoxin A by each BCA after 7 days. The probability values in bold were significant (p < 0.05). Results based on Fit Least Squares Method for the effects of water activity (a_w), temperature (T), CO₂ level, and BCA.

Factors	DF	p-Value (Prob > F)	Factors	DF	p-Value (Prob > F)
		OTA (µg/kg)			OTA (µg/kg)
BCA: Y1
aw	1	<0.0001	T* CO2	2	0.4169
T	2	0.0625	T*aw	2	0.0179
CO2	1	0.6508	CO2*aw*T	2	0.0039
aw *CO2	1	0.3699
BCA: Y2
aw	1	0.0007	T* CO2	2	0.6580
T	2	0.3874	T*aw	2	0.7202
CO2	1	0.4482	CO2*aw*T	2	0.2504
aw *CO2	1	0.2782
BCA: Y4
aw	1	<0.0001	T* CO2	2	<0.0001
T	2	<0.0001	T*aw	2	0.2624
CO2	1	0.0074	CO2*aw*T	2	<0.0001
aw *CO2	1	<0.0001

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/toxins17030110/s1. Figure S1: Examples of the five different levels of growth used to assess the growth level of A. carbonarius on the coffee cherries and the inoculum and a_w conditions during the experiment; Table S1: Summary of the statistical analysis for the effect of interacting climate-related abiotic factors on Ochratoxin A production by A. carbonarius after 7 days; Table S2: Summary of the statistical analysis for the effect of interacting climate-related abiotic factors and BCA on Ochratoxin A % of reduction by A. carbonarius after 7 days.

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